The nef gene of HIV and SIV encodes a myristylated protein dispensable for viral replication in vitro. A clearly defined function of this protein in viral replication has not been established. In vivo studies analyzing infection of rhesus macaques with mutants of siv demonstrated that nef is critical for maintenance of high virus load and progression to Simian AIDS. We have found that nef of these viruses associates with a cellular serine kinase. This activity phosphorylates two proteins that co-immunoprecipitate with nef in in vitro kinase assays. To identify the region(s) of nef important for association with cellular kinase activity, various nef alleles and mutants have been analyzed in the kinase assay. Two domains of nef are critical for association with the cellular serine kinase. The first domain is a membrane targeting signal at the N terminus; the second encompasses a central, highly conserved region found in all primate lentivirus nef genes. To determine whether the association of nef with the cellular kinase is essential for nef function in vivo, a kinase-negative point mutation was introduced into the pathogenic molecular clone SIVMAC39. In the mutant, arg137arg138 have been changed to leu137leu138. This mutant nef also does not downregulate cell surface CD4 antigen. Juvenile macaques were inoculated with the mutant virus to analyze the significance of the kinase association function in vivo. During the first four weeks of infection, relatively low virus loads were observed, but increased after eight. Virus recovered at 4 and 8 weeks after inoculation was composed of variants in which the nef gene sequence reverted to the prototype sequence, i.E., codons for leu137leu138 had mutated back to arg137arg138. The revertant nefs were able to associate with the cellular serine kinase and downregulated cell surface cd4 antigen. These data demonstrate a strong selective pressure for the reversion of nef from a kinase-negative phenotype to a kinase-positive phenotype in vivo. This reversion is directly linked to increased viral burden in the infected host.